Modified granulocyte macrophage colony stimulating factor (gm-csf) with reduced immunogenicity

ABSTRACT

The present invention relates to polypeptides to be administered especially to humans and in particular for therapeutic use. The polypeptides are modified polypeptides whereby the modification results in a reduced propensity for the polypeptide to elicit an immune response upon administration to the human subject. The invention in particular relates to the modification of human granulocyte macrophage colony stimulating factor (GM-CSF) to result in GM-CSF proteins that are substantially non-immunogenic or less immunogenic than any non-modified counterpart when used in vivo.

FIELD OF THE INVENTION

[0001] The present invention relates to polypeptides to be administeredespecially to humans and in particular for therapeutic use. Thepolypeptides are modified polypeptides whereby the modification resultsin a reduced propensity for the polypeptide to elicit an immune responseupon administration to the human subject. The invention in particularrelates to the modification of human granulocyte macrophage colonystimulating factor (GM-CSF) to result in GM-CSF protein variants thatare substantially non-immunogenic or less immunogenic than anynon-modified counterpart when used in vivo. The invention relatesfurthermore to T-cell epitope peptides derived from said non-modifiedprotein by means of which it is possible to create modified GM-CSFvariants with reduced immunogenicity.

BACKGROUND OF THE INVENTION

[0002] There are many instances whereby the efficacy of a therapeuticprotein is limited by an unwanted immune reaction to the therapeuticprotein. Several mouse monoclonal antibodies have shown promise astherapies in a number of human disease settings but in certain caseshave failed due to the induction of significant degrees of a humananti-murine antibody (HAMA) response [Schroff, R. W. et al (1985) CancerRes. 45: 879-885; Shawler, D. L. et al (1985) J. Immunol. 135:1530-1535]. For monoclonal antibodies, a number of techniques have beendeveloped in attempt to reduce the HAMA response [WO 89/09622; EP0239400; EP 0438310; WO 91/06667]. These recombinant DNA approaches havegenerally reduced the mouse genetic information in the final antibodyconstruct whilst increasing the human genetic information in the finalconstruct. Notwithstanding, the resultant “humanized” antibodies have,in several cases, still elicited an immune response in patients [IssacsJ. D. (1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999)Transplantation 68: 1417-1420].

[0003] Antibodies are not the only class of polypeptide moleculeadministered as a therapeutic agent against which an immune response maybe mounted. Even proteins of human origin and with the same amino acidsequences as occur within humans can still induce an immune response inhumans. Notable examples include the therapeutic use ofgranulocyte-macrophage colony stimulating factor [Wadhwa, M. et al(1999) Clin. Cancer Res. 5: 1353-1361] and interferon alpha 2 [Russo, D.et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl.J. Med. 318: 1409-1413] amongst others.

[0004] A principal factor in the induction of an immune response is thepresence within the protein of peptides that can stimulate the activityof T-cells via presentation on SMC class II molecules, so-called “T-cellepitopes”. Such potential T-cell epitopes are commonly defined as anyamino acid residue sequence with the ability to bind to MHC Class IImolecules. Such T-cell epitopes can be measured to establish MHCbinding. Implicitly, a “T-cell epitope” means an epitope which whenbound to MHC molecules can be recognized by a T-cell receptor (TCR), andwhich can, at least in principle, cause the activation of these T-cellsby engaging a TCR to promote a T-cell response. It is, however, usuallyunderstood that certain peptides which are found to bind to MHC Class IImolecules may be retained in a protein sequence because such peptidesare recognized as “self” within the organism into which the finalprotein is administered.

[0005] It is known, that certain of these T-cell epitope peptides can bereleased during the degradation of peptides, polypeptides or proteinswithin cells and subsequently be presented by molecules of the majorhistocompatability complex (MHC) in order to trigger the activation ofT-cells. For peptides presented by MHC Class II, such activation ofT-cells can then give rise, for example, to an antibody response bydirect stimulation of B-cells to produce such antibodies.

[0006] MHC Class II molecules are a group of highly polymorphic proteinswhich play a central role in helper T-cell selection and activation. Thehuman leukocyte antigen group DR (HLA-DR) are the predominant isotype ofthis group of proteins and are the major focus of the present invention.However, isotypes HLA-DQ and HLA-DP perform similar functions, hence thepresent invention is equally applicable to these. The MHC class II DRmolecule is made of an alpha and a beta chain which insert at theirC-termini through the cell membrane. Each hetero-dimer possesses aligand binding domain which binds to peptides varying between 9 and 20amino acids in length, although the binding groove can accommodate amaximum of 11 amino acids. The ligand binding domain is comprised ofamino acids 1 to 85 of the alpha chain, and amino acids 1 to 94 of thebeta chain. DQ molecules have recently been shown to have an homologousstructure and the DP family proteins are also expected to be verysimilar. In humans approximately 70 different allotypes of the DRisotype are known, for DQ there are 30 different allotypes and for DP 47different allotypes are known. Each individual bears two to four DRalleles, two DQ and two DP alleles. The structure of a number of DRmolecules has been solved and such structures point to an open-endedpeptide binding groove with a number of hydrophobic pockets which engagehydrophobic residues (pocket residues) of the peptide [Brown et alNature (1993) 364: 33; Stern et al (1994) Nature 368: 215]. Polymorphismidentifying the different allotypes of class II molecule contributes toa wide diversity of different binding surfaces for peptides within thepeptide binding grove and at the population level ensures maximalflexibility with regard to the ability to recognize foreign proteins andmount an immune response to pathogenic organisms. There is aconsiderable amount of polymorphism within the ligand binding domainwith distinct “families” within different geographical populations andethnic groups. This polymorphism affects the binding characteristics ofthe peptide binding domain, thus different “families” of DR moleculeswill have specificities for peptides with different sequence properties,although there may be some overlap. This specificity determinesrecognition of Th-cell epitopes (Class II T-cell response) which areultimately responsible for driving the antibody response to β-cellepitopes present on the same protein from which the Th-cell epitope isderived. Thus, the immune response to a protein in an individual isheavily influenced by T-cell epitope recognition which is a function ofthe peptide binding specificity of that individual's HLA-DR allotype.Therefore, in order to identify T-cell epitopes within a protein orpeptide in the context of a global population, it is desirable toconsider the binding properties of as diverse a set of HLA-DR allotypesas possible, thus covering as high a percentage of the world populationas possible.

[0007] An immune response to a therapeutic protein such as the proteinwhich is object of this invention, proceeds via the MHC class II peptidepresentation pathway. Here exogenous proteins are engulfed and processedfor presentation in association with MHC class II molecules of the DR,DQ or DP type. MHC Class II molecules are expressed by professionalantigen presenting cells (APCs), such as macrophages and dendritic cellsamongst others. Engagement of a MHC class II peptide complex by acognate T-cell receptor on the surface of the T-cell, together with thecross-binding of certain other co-receptors such as the CD4 molecule,can induce an activated state within the T-cell. Activation leads to therelease of cytokines further activating other lymphocytes such as Bcells to produce antibodies or activating T killer cells as a fullcellular immune response. The ability of a peptide to bind a given MHCclass II molecule for presentation on the surface of an APC is dependenton a number of factors most notably its primary sequence. This willinfluence both its propensity for proteolytic cleavage and also itsaffinity for binding within the peptide binding cleft of the MHC classII molecule. The MHC class II/peptide complex on the APC surfacepresents a binding face to a particular T-cell receptor (TCR) able torecognize determinants provided both by exposed residues of the peptideand the MHC class II molecule.

[0008] In the art there are procedures for identifying syntheticpeptides able to bind MHC class II molecules (e.g. WO98/52976 andWO00/34317). Such peptides may not function as T-cell epitopes in allsituations, particularly, in vivo due to the processing pathways orother phenomena. T-cell epitope identification is the first step toepitope elimination. The identification and removal of potential T-cellepitopes from proteins has been previously disclosed. In the art methodshave been provided to enable the detection of T-cell epitopes usually bycomputational means scanning for recognized sequence motifs inexperimentally determined T-cell epitopes or alternatively usingcomputational techniques to predict MHC class II-binding peptides and inparticular DR-binding peptides. WO98/52976 and WO00/34317 teachcomputational threading approaches to identifying polypeptide sequenceswith the potential to bind a sub-set of human MHC class II DR allotypes.In these teachings, predicted T-cell epitopes are removed by the use ofjudicious amino acid substitution within the primary sequence of thetherapeutic antibody or non-antibody protein of both non-human and humanderivation.

[0009] Other techniques exploiting soluble complexes of recombinant MHCmolecules in combination with synthetic peptides and able to bind toT-cell clones from peripheral blood samples from human or experimentalanimal subjects have been used in the art [Kern, F. et al (1998) NatureMedicine 4:975-978; Kwok, W. W. et al (2001) TRENDS in Immunology 22:583-588] and may also be exploited in an epitope identificationstrategy.

[0010] As depicted above and as consequence thereof, it would bedesirable to identify and to remove or at least to reduce T-cellepitopes from a given in principal therapeutically valuable butoriginally immunogenic peptide, polypeptide or protein.

[0011] One of these therapeutically valuable molecules is humangranulocyte macrophage colony stimulating factor (GM-CSF). GM-CSF is anacidic glycoprotein originally defined as stimulating the production ofgranulocytes and monocytes from their bone marrow precursors. Theprotein comprises 127 amino acid residues and shares significantsequence homology with GM-CSF proteins from other mammalian sources. Theavailability of recombinant GM-CSF has shown that this importanthaemopoietic growth factor is also able to stimulate the formation oferythroid and megakaryocyte precursors and in addition stimulates matureneutrophils, monocytes, eosinophils and mast cells to becomefunctionally activated [Kaushanski, K. et al (1986) Proc. Natl. Acad.Sci. U.S.A. 83: 3101; Emerson, S. G. et al (1985) Eur. J. Biochem.165:7; Weisbart, R. H. et al (1985) Nature 314: 361; Grabstein, K. H. et al(1985) Science 232:506].

[0012] The primary translation product of the human GM-CSF gene is aprotein of 144 amino acid residues with a mature (processed) form of 127residues. There is a high degree of homology between GM-CSF proteinsfrom other mammalian species and complete conservation of thedi-sulphide structure although GM-CSF from other species such as mouseare not able to stimulate human cells.

[0013] Others have provided GM-CSF molecules and analogues [U.S. Pat.No. 5,391,485; U.S. Pat. No. 5,032,676; U.S. Pat. No. 5,942,253; U.S.Pat. No. 6,120,807] but none of these teachings recognise the importanceof T cell epitopes to the immunogenic properties of the protein nor havebeen conceived to directly influence said properties in a specific andcontrolled way according to the scheme of the present invention.

[0014] The primary 127 amino acid sequence of human GM-CSF is asfollows:

[0015] APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE

[0016] However, there is a continued need for GM-CSF analogues withenhanced properties. Desired enhancements include alternative schemesand modalities for the expression and purification of the saidtherapeutic, but also and especially, improvements in the biologicalproperties of the protein. There is a particular need for enhancement ofthe in vivo characteristics when administered to the human subject. Inthis regard, it is highly desired to provide GM-CSF with reduced orabsent potential to induce an immune response in the human subject.

SUMMARY AND DESCRIPTION OF THE INVENTION

[0017] The present invention provides for modified forms of humangranulocyte macrophage colony stimulating factor (GM-CSF), in which theimmune characteristic is modified by means of reduced or removed numbersof potential T-cell epitopes.

[0018] The invention discloses sequences identified within the GM-CSFprimary sequence that are potential T-cell epitopes by virtue of MHCclass II binding potential. This disclosure specifically pertains thehuman GM-CSF protein being 127 amino acid residues. The inventiondiscloses also specific positions within the primary sequence of themolecule which according to the invention are to be altered by specificamino acid substitution, addition or deletion without in principalaffecting the biological activity. In cases in which the loss ofimmunogenicity can be achieved only by a simultaneous loss of biologicalactivity it is possible to restore said activity by further alterationswithin the amino acid sequence of the protein.

[0019] The invention furthermore discloses methods to produce suchmodified molecules, and above all methods to identify said T-cellepitopes which require alteration in order to reduce or removeimmunogenic sites.

[0020] The protein according to this invention would expect to displayan increased circulation time within the human subject and would be ofparticular benefit in chronic or recurring disease settings such as isthe case for a number of indications for GM-CSF. The present inventionprovides for modified forms of GM-CSF proteins that are expected todisplay enhanced properties in vivo. These modified GM-CSP molecules canbe used in pharmaceutical compositions.

[0021] In summary the invention relates to the following issues:

[0022] a modified molecule having the biological activity of humanGM-CSF and being substantially non-immunogenic or less immunogenic thanany non-modified molecule having the same biological activity when usedin vivo;

[0023] an accordingly specified molecule, wherein said loss ofimmunogenicity is achieved by removing one or more T-cell epitopesderived from the originally non-modified molecule;

[0024] an accordingly specified molecule, wherein said loss ofimmunogenicity is achieved by reduction in numbers of MHC allotypes ableto bind peptides derived from said molecule;

[0025] an accordingly specified molecule, wherein one T-cell epitope isremoved;

[0026] an accordingly specified molecule, wherein said originallypresent T-cell epitopes are MHC class II ligands or peptide sequenceswhich show the ability to stimulate or bind T-cells via presentation onclass II;

[0027] an accordingly specified molecule, wherein said peptide sequencesare selected from the group as depicted in Table 1;

[0028] an accordingly specified molecule, wherein 1-9 amino acidresidues, preferably one amino acid residue in any of the originallypresent T-cell epitopes are altered;

[0029] an accordingly specified molecule, wherein the alteration of theamino acid residues is substitution, addition or deletion of originallypresent amino acid(s) residue(s) by other amino acid residue(s) atspecific position(s);

[0030] an accordingly specified molecule, wherein one or more of theamino acid residue substitutions are carried out as indicated in Table2;

[0031] an accordingly specified molecule, wherein (additionally) one ormore of the amino acid residue substitutions are carried out asindicated in Table 3 for the reduction in the number of MHC allotypesable to bind peptides derived from said molecule;

[0032] an accordingly specified molecule, wherein, if necessary,additionally further alteration usually by substitution, addition ordeletion of specific amino acid(s) is conducted to restore biologicalactivity of said molecule;

[0033] a DNA sequence or molecule which codes for any of said specifiedmodified molecules as defined above and below;

[0034] a pharmaceutical composition comprising a modified moleculehaving the biological activity of GM-CSF as defined above and/or in theclaims, optionally together with a pharmaceutically acceptable carrier,diluent or excipient;

[0035] a method for manufacturing a modified molecule having thebiological activity of GM-CSF as defined in any of the claims of theabove-cited claims comprising the following steps: (i) determining theamino acid sequence of the polypeptide or part thereof; (ii) identifyingone or more potential T-cell epitopes within the amino acid sequence ofthe protein by any method including determination of the binding of thepeptides to MHC molecules using in vitro or in silico techniques orbiological assays; (iii) designing new sequence variants with one ormore amino acids within the identified potential T-cell epitopesmodified in such a way to substantially reduce or eliminate the activityof the T-cell epitope as determined by the binding of the peptides toMHC molecules using in vitro or in silico techniques or biologicalassays; (iv) constructing such sequence variants by recombinant DNAtechniques and testing said variants in order to identify one or morevariants with desirable properties; and (v) optionally repeating steps(ii)-(iv);

[0036] an accordingly specified method, wherein step (iii) is carriedout by substitution, addition or deletion of 1-9 amino acid residues inany of the originally present T-cell epitopes;

[0037] an accordingly specified method, wherein the alteration is madewith reference to an homologous protein sequence and/or in silicomodeling techniques;

[0038] an accordingly specified method, wherein step (ii) of above iscarried out by the following steps: (a) selecting a region of thepeptide having a known amino acid residue sequence; (b) sequentiallysampling overlapping amino acid residue segments of predetermineduniform size and constituted by at least three amino acid residues fromthe selected region; (c) calculating MHC Class II molecule binding scorefor each said sampled segment by summing assigned values for eachhydrophobic amino acid residue side chain present in said sampled aminoacid residue segment; and (d) identifying at least one of said segmentssuitable for modification, based on the calculated MHC Class II moleculebinding score for that segment, to change overall MHC Class II bindingscore for the peptide without substantially reducing therapeutic utilityof the peptide; step (c) is preferably carried out by using a Böhmscoring function modified to include 12-6 van der Waal's ligand-proteinenergy repulsive term and ligand conformational energy term by (1)providing a first data base of MHC Class II molecule models; (2)providing a second data base of allowed peptide backbones for said MHCClass II molecule models; (3) selecting a model from said first database; (4) selecting an allowed peptide backbone from said second database; (5) identifying amino acid residue side chains present in eachsampled segment; (6) determining the binding affinity value for all sidechains present in each sampled segment; and repeating steps (1) through(5) for each said model and each said backbone;

[0039] a 13mer T-cell epitope peptide having a potential MHC class IIbinding activity and created from immunogenetically non-modified GM-CSF,selected from the group as depicted in Table 1 and its use for themanufacture of GM-CSF having substantially no or less immunogenicitythan any non-modified molecule with the same biological activity whenused in vivo;

[0040] a peptide sequence consisting of at least 9 consecutive aminoacid residues of a 13mer T-cell epitope peptide as specified above andits use for the manufacture of GM-CSF having substantially no or lessimmunogenicity than any non-modified molecule with the same biologicalactivity when used in vivo;

[0041] an immunogenicly modified molecule having the biological activityof human GM-CSF obtainable by any of the methods as specified above andbelow.

[0042] The term “T-cell epitope” means according to the understanding ofthis invention an amino acid sequence which is able to bind MHC classII, able to stimulate T-cells and/or also to bind (without necessarilymeasurably activating) T-cells in complex with MHC class II. The term“peptide” as used herein and in the appended claims, is a compound thatincludes two or more amino acids. The amino acids are linked together bya peptide bond (defined herein below). There are 20 different naturallyoccurring amino acids involved in the biological production of peptides,and any number of them may be linked in any order to form a peptidechain or ring. The naturally occurring amino acids employed in thebiological production of peptides all have the L-configuration.Synthetic peptides can be prepared employing conventional syntheticmethods, utilizing L-amino acids, D-amino acids, or various combinationsof amino acids of the two different configurations. Some peptidescontain only a few amino acid units. Short peptides, e.g., having lessthan ten amino acid units, are sometimes referred to as “oligopeptides”.Other peptides contain a large number of amino acid residues, e.g. up to100 or more, and are referred to as “polypeptides”. By convention, a“polypeptide” may be considered as any peptide chain containing three ormore amino acids, whereas a “oligopeptide” is usually considered as aparticular type of “short” polypeptide. Thus, as used herein, it isunderstood that any reference to a “polypeptide” also includes anoligopeptide. Further, any reference to a “peptide” includespolypeptides, oligopeptides, and proteins. Each different arrangement ofamino acids forms different polypeptides or proteins. The number ofpolypeptides—and hence the number of different proteins—that can beformed is practically unlimited. “Alpha carbon (Cα)” is the carbon atomof the carbon-hydrogen (CH) component that is in the peptide chain. A“side chain” is a pendant group to Cα that can comprise a simple orcomplex group or moiety, having physical dimensions that can varysignificantly compared to the dimensions of the peptide.

[0043] The invention may be applied to any GM-CSF species of moleculewith substantially the same primary amino acid sequences as thosedisclosed herein and would include therefore GM-CSF molecules derived bygenetic engineering means or other processes and may contain more orless than 127 amino acid residues.

[0044] M-CSF proteins such as identified from other mammalian sourceshave in common many of the peptide sequences of the present disclosureand have in common many peptide sequences with substantially the samesequence as those of the disclosed listing. Such protein sequencesequally therefore fall under the scope of the present invention.

[0045] The invention is conceived to overcome the practical reality thatsoluble proteins introduced into autologous organisms can trigger animmune response resulting in development of host antibodies that bind tothe soluble protein. One example amongst others, is interferon alpha 2to which a proportion of human patients make antibodies despite the factthat this protein is produced endogenously [Russo, D. et al (1996) ibid;Stein, R. et al (1988) ibid]. It is likely that the same situationpertains to the therapeutic use of GM-CSF and the present inventionseeks to address this by providing GM-CSF proteins with alteredpropensity to elicit an immune response on administration to the humanhost.

[0046] The general method of the present invention leading to themodified GM-CSF comprises the following steps:

[0047] (a) determining the amino acid sequence of the polypeptide orpart thereof;

[0048] (b) identifying one or more potential T-cell epitopes within theamino acid sequence of the protein by any method including determinationof the binding of the peptides to MHC molecules using in vitro or insilico techniques or biological assays;

[0049] (c) designing new sequence variants with one or more amino acidswithin the identified potential T-cell epitopes modified in such a wayto substantially reduce or eliminate the activity of the T-cell epitopeas determined by the binding of the peptides to MHC molecules using invitro or in silico techniques or biological assays. Such sequencevariants are created in such a way to avoid creation of new potentialT-cell epitopes by the sequence variations unless such new potentialT-cell epitopes are, in turn, modified in such a way to substantiallyreduce or eliminate the activity of the T-cell epitope; and

[0050] (d) constructing such sequence variants by recombinant DNAtechniques and testing said variants in order to identify one or morevariants with desirable properties according to well known recombinanttechniques.

[0051] The identification of potential T-cell epitopes according to step(b) can be carried out according to methods describes previously in theprior art. Suitable methods are disclosed in WO 98/59244; WO 98/52976;WO 00/34317 and may preferably be used to identify binding propensity ofGM-CSF-derived peptides to an MHC class II molecule.

[0052] Another very efficacious method for identifying T-cell epitopesby calculation is described in the EXAMPLE which is a preferredembodiment according to this invention.

[0053] In practice a number of variant GM-CSF proteins will be producedand tested for the desired immune and functional characteristic. Thevariant proteins will most preferably be produced by recombinant DNAtechniques although other procedures including chemical synthesis ofGM-CSF fragments may be contemplated.

[0054] The results of an analysis according to step (b) of the abovescheme and pertaining to the human GM-CSF protein sequence 127 aminoacid residues is presented in Table 1. TABLE 1 Peptide sequences inhuman GM-CSF with potential human MHC class II binding activity.PSPSTQPWEHVNA, QPWEHVNAIQEAR, EHVNAIQEARRLL, HVNAIQEARRLLN,VNAIQEARRLLNL, NAIQEARRLLNLS, RRLLNLSRDTAAE, RLLNLSRDTAAEM,LNLSRDTAAEMNE, RDTAAEMNETVEV, AEMNETVEVISEM, NETVEVISEMFDL,ETVEVISEMFDLQ, VEVISEMFDLQEP, EVISEMFDLQEPT, ISEMFDLQEPTCL,SEMFDLQEPTCLQ, EMFDLQEPTCLQT, MFDLQEPTCLQTR, FDLQEPTCLQTRL,EPTCLQTRLELYK, TCLQTRLELYKQG, QTRLELYKQGLRG, TRLELYKQGLRGS,LELYKQGLRGSLT, ELYKQGLRGSLTK, QGLRGSLTKLKGP, RGSLTKLKGPLTM,GSLTKLKGPLTMM, SLTKLKGPLTMMA, TKLKGPLTMMASH, KGPLTMMASHYKQ,GPLTMMASHYKQH, PLTMMASHYKQHC, LTMMASHYKQHCP, TMMASHYKQHCPP,SHYKQHCPPTPET, CPPTPETSCATQT, PETSCATQTITFE, CATQTITFESFKE,QTITFESFKENLK, ITFESSKENLKDF, ESFKENLKDFLLV, SFKENLKDFLLVI,ENLKDFLLVIPFD, NLKDFLLVIPFDC, KDFLLVIPFDCWE, DFLLVIPFDCWEP,LLVIPFDCWEPVQ

[0055] Peptides are 13mers, amino acids are identified using singleletter code.

[0056] The results of a design and constructs according to step (c) and(d) of the above scheme and pertaining to the modified molecule of thisinvention is presented in Tables 2 and 3. TABLE 2 Substitutions leadingto the elimination of potential T-cell epitopes of human GM-CSF (WT =wild type). Residue WT # Residue Substitution 16 V A C D E G H K N P Q RS T 19 I A C D E G H K N P Q R S T 25 L A C D E G H K N P Q R S T 26 L AC D E G H K N P Q R S T 28 L A C D E F G H K N P Q R S T 36 M A C D E GH K N P Q R S T 40 V A C D E G H K N P Q R S T 43 I A C D E G H K N P QR S T 46 M A C D E G H K N P Q R S T 47 F A C D E G H K N P Q R S T 49 LA C D E G H K N P Q R S T 55 L A C D E G H K N P Q R S T 59 L A C D E GH K N P Q R S T 61 L A C D E G H K N P Q R S T 62 Y A C D E G H K N P QR S T 66 L A C D E G H K N P Q R S T 70 L A C D E G H K N P Q R S T 73 LA C D E G H K N P Q R S T 77 L A C D E G H K N P Q R S T 79 M A C D E GH K N P Q R S T 80 M A C D E G H K N P Q R S T 84 Y C D E G H N P R S T101 I A C D E G H K N P Q R S T 106 F A C D E G H K N P Q R S T 110 L AC D E G H K N P Q R S T 113 F A C D E G H K N P Q R S T 114 L A C D E GH K N P Q R S T 115 L A C D E G H K N P Q R S T 117 I A C D E G H K N PQ R S T

[0057] TABLE 3 Additional substitutions leading to the removal of apotential T-cell epitope for 1 or more MHC allotypes. Residue WT #Residue Substitution 15 H A C F G I L M P V W Y 18 A F H K L N P Q R S TW Y 20 Q T 21 E F I P V W Y 22 A D E F H I K N P Q R S T V W 24 R A C FG I L M P V W Y 26 L F I M V W Y 31 D H 34 A H K N P Q R S T V W Y 35 EA C G P 36 M W Y 37 N A C G P 38 E A C G P 42 V A C D E G H K M N P Q RS T W 45 E A C F G I L M P V W Y 47 F W 49 L W Y 50 Q P 60 E A C G P 61L F I M 63 K A C G I M P Y 64 Q A C G P 66 L F I M V 67 R A C G P 69 S T70 L M W 71 T A C G P 72 K T 74 K T 75 G H P 77 L F I W Y 78 T A C G P WY 82 S A C F G M P V W Y 85 K H P 87 H A C F G I M P W Y 88 C D E H K NP Q R S T W 109  N T 121  C P Y 122  W T

[0058] The invention relates to GM-CSF analogues in which substitutionsof at least one amino acid residue have been made at positions resultingin a substantial reduction in activity of or elimination of one or morepotential T-cell epitopes from the protein. One or more amino acidsubstitutions at particular points within any of the potential MHC classII ligands identified in Table 1 may result in a GM-CSF molecule with areduced immunogenic potential when administered as a therapeutic to thehuman host. Preferably, amino acid substitutions are made at appropriatepoints within the peptide sequence predicted to achieve substantialreduction or elimination of the activity of the T-cell epitope. Inpractice an appropriate point will preferably equate to an amino acidresidue binding within one of the pockets provided within the MHC classII binding groove.

[0059] It is most preferred to alter binding within the first pocket ofthe cleft at the so-called P1 or P1 anchor position of the peptide. Thequality of binding interaction between the P1 anchor residue of thepeptide and the first pocket of the MHC class II binding groove isrecognized as being a major determinant of overall binding affinity forthe whole peptide. An appropriate substitution at this position of thepeptide will be for a residue less readily accommodated within thepocket, for example, substitution to a more hydrophilic residue. Aminoacid residues in the peptide at positions equating to binding withinother pocket regions within the MHC binding cleft are also consideredand fall under the scope of the present.

[0060] It is understood that single amino acid substitutions within agiven potential T-cell epitope are the most preferred route by which theepitope may be eliminated. Combinations of substitution within a singleepitope may be contemplated and for example can be particularlyappropriate where individually defined epitopes are in overlap with eachother. Moreover, amino acid substitutions either singly within a givenepitope or in combination within a single epitope may be made atpositions not equating to the “pocket residues” with respect to the MHCclass II binding groove, but at any point within the peptide sequence.Substitutions may be made with reference to an homologues structure orstructural method produced using in silico techniques known in the artand may be based on known structural features of the molecule accordingto this invention. All such substitutions fall within the scope of thepresent invention.

[0061] Amino acid substitutions other than within the peptidesidentified above may be contemplated particularly when made incombination with substitution(s) made within a listed peptide. Forexample a change may be contemplated to restore structure or biologicalactivity of the variant molecule. Such compensatory changes and changesto include deletion or addition of particular amino acid residues fromthe GM-CSF polypeptide resulting in a variant with desired activity andin combination with changes in any of the disclosed peptides fall underthe scope of the present.

[0062] In as far as this invention relates to modified GM-CSF,compositions containing such modified GM-CSF proteins or fragments ofmodified GM-CSF proteins and related compositions should be consideredwithin the scope of the invention. In another aspect, the presentinvention relates to nucleic acids encoding modified GM-CSF entities. Ina further aspect the present invention relates to methods fortherapeutic treatment of humans using the modified GM-CSF proteins.

EXAMPLE

[0063] There are a number of factors that play important roles indetermining the total structure of a protein or polypeptide. First, thepeptide bond, i.e., that bond which joins the amino acids in the chaintogether, is a covalent bond. This bond is planar in structure,essentially a substituted amide. An “amide” is any of a group of organiccompounds containing the grouping —CONH—.

[0064] The planar peptide bond linking Cα of adjacent amino acids may berepresented as depicted below:

[0065] Because the O═C and the C—N atoms lie in a relatively rigidplane, free rotation does not occur about these axes. Hence, a planeschematically depicted by the interrupted line is sometimes referred toas an “amide” or “peptide plane” plane wherein lie the oxygen (O),carbon (C), nitrogen (N), and hydrogen (H) atoms of the peptidebackbone. At opposite corners of this amide plane are located the Cαatoms. Since there is substantially no rotation about the O═C and C—Natoms in the peptide or amide plane, a polypeptide chain thus comprisesa series of planar peptide linkages joining the Cα atoms.

[0066] A second factor that plays an important role in defining thetotal structure or conformation of a polypeptide or protein is the angleof rotation of each amide plane about the common Cα linkage. The terms“angle of rotation” and “torsion angle” are hereinafter regarded asequivalent terms. Assuming that the O, C, N, and H atoms remain in theamide plane (which is usually a valid assumption, although there may besome slight deviations from planarity of these atoms for someconformations), these angles of rotation define the N and Rpolypeptide's backbone conformation, i.e., the structure as it existsbetween adjacent residues. These two angles are known as φ and ψ. A setof the angles φ₁, ψ₁, where the subscript i represents a particularresidue of a polypeptide chain, thus effectively defines the polypeptidesecondary structure. The conventions used in defining the φ, ψ angles,i.e., the reference points at which the amide planes form a zero degreeangle, and the definition of which angle is φ, and which angle is ψ, fora given polypeptide, are defined in the literature. See, e.g.,Ramachandran et al. Adv. Prot. Chem. 23:283-437 (1968), at pages 285-94,which pages are incorporated herein by reference.

[0067] The present method can be applied to any protein, and is based inpart upon the discovery that in humans the primary Pocket 1 anchorposition of MHC Class II molecule binding grooves has a well designedspecificity for particular amino acid side chains. The specificity ofthis pocket is determined by the identity of the amino acid at position86 of the beta chain of the MHC Class II molecule. This site is locatedat the bottom of Pocket 1 and determines the size of the side chain thatcan be accommodated by this pocket. Marshall, K. W., J. Immunol.,152:4946-4956 (1994). If this residue is a glycine, then all hydrophobicaliphatic and aromatic amino acids (hydrophobic aliphatics being:valine, leucine, isoleucine, methionine and aromatics being:phenylalanine, tyrosine and tryptophan) can be accommodated in thepocket, a preference being for the aromatic side chains. If this pocketresidue is a valine, then the side chain of this amino acid protrudesinto the pocket and restricts the size of peptide side chains that canbe accommodated such that only hydrophobic aliphatic side chains can beaccommodated. Therefore, in an amino acid residue sequence, wherever anamino acid with a hydrophobic aliphatic or aromatic side chain is found,there is the potential for a MHC Class II restricted T-cell epitope tobe present. If the side-chain is hydrophobic aliphatic, however, it isapproximately twice as, likely to be associated with a T-cell epitopethan an aromatic side chain (assuming an approximately even distributionof Pocket 1 types throughout the global population).

[0068] A computational method embodying the present invention profilesthe likelihood of peptide regions to contain T-cell epitopes as follows:

[0069] (1) The primary sequence of a peptide segment of predeterminedlength is scanned, and all hydrophobic aliphatic and aromatic sidechains present are identified. (2)The hydrophobic aliphatic side chainsare assigned a value greater than that for the aromatic side chains;preferably about twice the value assigned to the aromatic side chains,e.g., a value of 2 for a hydrophobic aliphatic side chain and a value of1 for an aromatic side chain. (3) The values determined to be presentare summed for each overlapping amino acid residue segment (window) ofpredetermined uniform length within the peptide, and the total value fora particular segment (window) is assigned to a single amino acid residueat an intermediate position of the segment (window), preferably to aresidue at about the midpoint of the sampled segment (window). Thisprocedure is repeated for each sampled overlapping amino acid residuesegment (window). Thus, each amino acid residue of the peptide isassigned a value that relates to the likelihood of a T-cell epitopebeing present in that particular segment (window). (4) The valuescalculated and assigned as described in Step 3, above, can be plottedagainst the amino acid coordinates of the entire amino acid residuesequence being assessed. (5) All portions of the sequence which have ascore of a predetermined value, e.g., a value of 1, are deemed likely tocontain a T-cell epitope and can be modified, if desired.

[0070] This particular aspect of the present invention provides ageneral method by which the regions of peptides likely to contain T-cellepitopes can be described. Modifications to the peptide in these regionshave the potential to modify the MHC Class II binding characteristics.

[0071] According to another aspect of the present invention, T-cellepitopes can be predicted with greater accuracy by the use of a moresophisticated computational method which takes into account theinteractions of peptides with models of MHC Class II alleles. Thecomputational prediction of T-cell epitopes present within a peptideaccording to this particular aspect contemplates the construction ofmodels of at least 42 MHC Class II alleles based upon the structures ofall known MHC Class II molecules and a method for the use of thesemodels in the computational identification of T-cell epitopes, theconstruction of libraries of peptide backbones for each model in orderto allow for the known variability in relative peptide backbone alphacarbon (Cα) positions, the construction of libraries of amino-acid sidechain conformations for each backbone dock with each model for each ofthe 20 amino-acid alternatives at positions critical for the interactionbetween peptide and MHC Class II molecule, and the use of theselibraries of backbones and side-chain conformations in conjunction witha scoring function to select the optimum backbone and side-chainconformation for a particular peptide docked with a particular MHC ClassII molecule and the derivation of a binding score from this interaction.

[0072] Models of MHC Class II molecules can be derived via homologymodeling from a number of similar structures found in the BrookhavenProtein Data Bank (“PDB”). These may be made by the use ofsemi-automatic homology modeling software (Modeller, Sali A. & BlundellT L., 1993. J. Mol Biol 234:779-815) which incorporates a simulatedannealing function, in conjunction with the CHARMm force-field forenergy minimisation (available from Molecular Simulations Inc., SanDiego, Calif.). Alternative modeling methods can be utilized as well.

[0073] The present method differs significantly from other computationalmethods which use libraries of experimentally derived binding data ofeach amino-acid alternative at each position in the binding groove for asmall set of MHC Class II molecules (Marshall, K. W., et al., Biomed.Pept. Proteins Nucleic Acids, 1(3):157-162) (1995) or yet othercomputational methods which use similar experimental binding data inorder to define the binding characteristics of particular types ofbinding pockets within the groove, again using a relatively small subsetof MHC Class II molecules, and then ‘mixing and matching’ pocket typesfrom this pocket library to artificially create further ‘virtual’ MHCClass II molecules (Sturniolo T., et al., Nat. Biotech, 17(6): 555-561(1999). Both prior methods suffer the major disadvantage that, due tothe complexity of the assays and the need to synthesize large numbers ofpeptide variants, only a small number of MHC Class II molecules can beexperimentally scanned. Therefore the first prior method can only makepredictions for a small number of MHC Class II molecules. The secondprior method also makes the assumption that a pocket lined with similaramino-acids in one molecule will have the same binding characteristicswhen in the context of a different Class II allele and suffers furtherdisadvantages in that only those MHC Class II molecules can be‘virtually’ created which contain pockets contained within the pocketlibrary. Using the modeling approach described herein, the structure ofany number and type of MHC Class II molecules can be deduced, thereforealleles can be specifically selected to be representative of the globalpopulation. In addition, the number of MHC Class II molecules scannedcan be increased by making further models further than having togenerate additional data via complex experimentation.

[0074] The use of a backbone library allows for variation in thepositions of the Cα atoms of the various peptides being scanned whendocked with particular MHC Class II molecules. This is again in contrastto the alternative prior computational methods described above whichrely on the use of simplified peptide backbones for scanning amino-acidbinding in particular pockets. These simplified backbones are not likelyto be representative of backbone conformations found in ‘real’ peptidesleading to inaccuracies in prediction of peptide binding. The presentbackbone library is created by superposing the backbones of all peptidesbound to MHC Class II molecules found within the Protein Data Bank andnoting the root mean square (RMS) deviation between the Cα atoms of eachof the eleven amino-acids located within the binding groove. While thislibrary can be derived from a small number of suitable available mouseand human structures (currently 13), in order to allow for thepossibility of even greater variability, the RMS figure for each C″-αposition is increased by 50%. The average Cα position of each amino-acidis then determined and a sphere drawn around this point whose radiusequals the RMS deviation at that position plus 50%. This sphererepresents all allowed Cα positions.

[0075] Working from the Cα with the least RMS deviation (that of theamino-acid in Pocket 1 as mentioned above, equivalent to Position 2 ofthe 11 residues in the binding groove), the sphere isthree-dimensionally gridded, and each vertex within the grid is thenused as a possible location for a Cα of that amino-acid. The subsequentamide plane, corresponding to the peptide bond to the subsequentamino-acid is grafted onto each of these Cαs and the φ and ψ angles arerotated step-wise at set intervals in order to position the subsequentCα. If the subsequent Cα falls within the ‘sphere of allowed positions’for this Cα than the orientation of the dipeptide is accepted, whereasif it falls outside the sphere then the dipeptide is rejected. Thisprocess is then repeated for each of the subsequent Cα positions, suchthat the peptide grows from the Pocket 1 Cα ‘seed’, until all ninesubsequent Cαs have been positioned from all possible permutations ofthe preceding Cαs. The process is then repeated once more for the singleCα preceding pocket 1 to create a library of backbone Cα positionslocated within the binding groove.

[0076] The number of backbones generated is dependent upon severalfactors: The size of the ‘spheres of allowed positions’; the fineness ofthe gridding of the ‘primary sphere’ at the Pocket 1 position; thefineness of the step-wise rotation of the φ and ψ angles used toposition subsequent Cαs. Using this process, a large library ofbackbones can be created. The larger the backbone library, the morelikely it will be that the optimum fit will be found for a particularpeptide within the binding groove of an MHC Class II molecule. Inasmuchas all backbones will not be suitable for docking with all the models ofMHC Class II molecules due to clashes with amino-acids of the bindingdomains, for each allele a subset of the library is created comprisingbackbones which can be accommodated by that allele. The use of thebackbone library, in conjunction with the models of MHC Class IImolecules creates an exhaustive database consisting of allowed sidechain conformations for each amino-acid in each position of the bindinggroove for each MHC Class II molecule docked with each allowed backbone.This data set is generated using a simple steric overlap function wherea MHC Class II molecule is docked with a backbone and an amino-acid sidechain is grafted onto the backbone at the desired position. Each of therotatable bonds of the side chain is rotated step-wise at set intervalsand the resultant positions of the atoms dependent upon that bond noted.The interaction of the atom with atoms of side-chains of the bindinggroove is noted and positions are either accepted or rejected accordingto the following criteria: The sum total of the overlap of all atoms sofar positioned must not exceed a pre-determined value. Thus thestringency of the conformational search is a function of the intervalused in the step-wise rotation of the bond and the pre-determined limitfor the total overlap. This latter value can be small if it is knownthat a particular pocket is rigid, however the stringency can be relaxedif the positions of pocket side-chains are known to be relativelyflexible. Thus allowances can be made to imitate variations inflexibility within pockets of the binding groove. This conformationalsearch is then repeated for every amino-acid at every position of eachbackbone when docked with each of the MHC Class II molecules to createthe exhaustive database of side-chain conformations.

[0077] A suitable mathematical expression is used to estimate the energyof binding between models of MHC Class II molecules in conjunction withpeptide ligand conformations which have to be empirically derived byscanning the large database of backbone/side-chain conformationsdescribed above. Thus a protein is scanned for potential T-cell epitopesby subjecting each possible peptide of length varying between 9 and 20amino-acids (although the length is kept constant for each scan) to thefollowing computations: An MHC Class II molecule is selected togetherwith a peptide backbone allowed for that molecule and the side-chainscorresponding to the desired peptide sequence are grafted on. Atomidentity and interatomic distance data relating to a particularside-chain at a particular position on the backbone are collected foreach allowed conformation of that amino-acid (obtained from the databasedescribed above). This is repeated for each side-chain along thebackbone and peptide scores derived using a scoring function. The bestscore for that backbone is retained and the process repeated for eachallowed backbone for the selected model. The scores from all allowedbackbones are compared and the highest score is deemed to be the peptidescore for the desired peptide in that MHC Class II model. This processis then repeated for each model with every possible peptide derived fromthe protein being scanned, and the scores for peptides versus models aredisplayed.

[0078] In the context of the present invention, each ligand presentedfor the binding affinity calculation is an amino-acid segment selectedfrom a peptide or protein as discussed above. Thus, the ligand is aselected stretch of amino acids about 9 to 20 amino acids in lengthderived from a peptide, polypeptide or protein of known sequence. Theterms “amino acids” and “residues” are hereinafter regarded asequivalent terms. The ligand, in the form of the consecutive amino acidsof the peptide to be examined grafted onto a backbone from the backbonelibrary, is positioned in the binding cleft of an MHC Class II moleculefrom the MHC Class II molecule model library via the coordinates of theC″-α atoms of the peptide backbone and an allowed conformation for eachside-chain is elected from the database of allowed conformations. Therelevant atom identities and interatomic distances are also retrievedfrom this database and used to calculate the peptide binding score.Ligands with a high binding affinity for the MHC Class II binding pocketare flagged as candidates for site-directed mutagenesis. Amino-acidsubstitutions are made in the flagged ligand (and hence in the proteinof interest) which is then retested using the scoring function in orderto determine changes which reduce the binding affinity below apredetermined threshold value. These changes can then be incorporatedinto the protein of interest to remove T-cell epitopes.

[0079] Binding between the peptide ligand and the binding groove of MHCClass II molecules involves non-covalent interactions including, but notlimited to: hydrogen bonds, electrostatic interactions, hydrophobic(lipophilic) interactions and Van der Walls interactions. These areincluded in the peptide scoring function as described in detail below.It should be understood that a hydrogen bond is a non-covalent bondwhich can be formed between polar or charged groups and consists of ahydrogen atom shared by two other atoms. The hydrogen of the hydrogendonor has a positive charge where the hydrogen acceptor has a partialnegative charge. For the purposes of peptide/protein interactions,hydrogen bond donors may be either nitrogens with hydrogen attached orhydrogens attached to oxygen or nitrogen. Hydrogen bond acceptor atomsmay be oxygens not attached to hydrogen, nitrogens with no hydrogensattached and one or two connections, or sulphurs with only oneconnection. Certain atoms, such as oxygens attached to hydrogens orimine nitrogens (e.g. C═NH) may be both hydrogen acceptors or donors.Hydrogen bond energies range from 3 to 7 Kcal/mol and are much strongerthan Van der Waal's bonds, but weaker than covalent bonds. Hydrogenbonds are also highly directional and are at their strongest when thedonor atom, hydrogen atom and acceptor atom are co-linear. Electrostaticbonds are formed between oppositely charged ion pairs and the strengthof the interaction is inversely proportional to the square of thedistance between the atoms according to Coulomb's law. The optimaldistance between ion pairs is about 2.8 Å. In protein/peptideinteractions, electrostatic bonds may be formed between arginine,histidine or lysine and aspartate or glutamate. The strength of the bondwill depend upon the pKa of the ionizing group and the dielectricconstant of the medium although they are approximately similar instrength to hydrogen bonds.

[0080] Lipophilic interactions are favorable hydrophobic-hydrophobiccontacts that occur between he protein and peptide ligand. Usually,these will occur between hydrophobic amino acid side chains of thepeptide buried within the pockets of the binding groove such that theyare not exposed to solvent. Exposure of the hydrophobic residues tosolvent is highly unfavorable since the surrounding solvent moleculesare forced to hydrogen bond with each other forming cage-like clathratestructures. The resultant decrease in entropy is highly unfavorable.Lipophilic atoms may be sulphurs which are neither polar nor hydrogenacceptors and carbon atoms which are not polar.

[0081] Van der Waal's bonds are non-specific forces found between atomswhich are 3-4 Å apart. They are weaker and less specific than hydrogenand electrostatic bonds. The distribution of electronic charge around anatom changes with time and, at any instant, the charge distribution isnot symmetric. This transient asymmetry in electronic charge induces asimilar asymmetry in neighboring atoms. The resultant attractive forcesbetween atoms reaches a maximum at the Van der Waal's contact distancebut diminishes very rapidly at about 1 Å to about 2 Å. Conversely, asatoms become separated by less than the contact distance, increasinglystrong repulsive forces become dominant as the outer electron clouds ofthe atoms overlap. Although the attractive forces are relatively weakcompared to electrostatic and hydrogen bonds (about 0.6 Kcal/mol), therepulsive forces in particular may be very important in determiningwhether a peptide ligand may bind successfully to a protein.

[0082] In one embodiment, the Böhm scoring function (SCORE1 approach) isused to estimate the binding constant. (Böhm, H. J., J. Comput AidedMol. Des., 8(3):243-256 (1994) which is hereby incorporated in itsentirety). In another embodiment, the scoring function (SCORE2 approach)is used to estimate the binding affinities as an indicator of a ligandcontaining a T-cell epitope (Böhm, H. J., J. Comput Aided Mol. Des.,12(4):309-323 (1998) which is hereby incorporated in its entirety).However, the Böhm scoring functions as described in the above referencesare used to estimate the binding affinity of a ligand to a protein whereit is already known that the ligand successfully binds to the proteinand the protein/ligand complex has had its structure solved, the solvedstructure being present in the Protein Data Bank (“PDB”). Therefore, thescoring function has been developed with the benefit of known positivebinding data. In order to allow for discrimination between positive andnegative binders, a repulsion term must be added to the equation. Inaddition, a more satisfactory estimate of binding energy is achieved bycomputing the lipophilic interactions in a pairwise manner rather thanusing the area based energy term of the above Böhm functions. Therefore,in a preferred embodiment, the binding energy is estimated using amodified Böhm scoring function. In the modified Böhm scoring function,the binding energy between protein and ligand (ΔG_(bind)) is estimatedconsidering the following parameters: The reduction of binding energydue to the overall loss of translational and rotational entropy of theligand (ΔG₀); contributions from ideal hydrogen bonds (ΔG_(hb)) where atleast one partner is neutral; contributions from unperturbed ionicinteractions (ΔG_(ionic)); lipophilic interactions between lipophilicligand atoms and lipophilic acceptor atoms (ΔG_(lipo)); the loss ofbinding energy due to the freezing of internal degrees of freedom in theligand, i.e., the freedom of rotation about each C—C bond is reduced(ΔG_(rot)); the energy of the interaction between the protein and ligand(E_(VdW)). Consideration of these terms gives equation 1:

(ΔG _(bind))=(ΔG ₀)+(ΔG _(hb) ×N _(hb))+(ΔG _(ionic) ×N _(ionic))+(ΔG_(lipo) ×N _(lipo))+(ΔG _(rot) +N _(rot))+(E _(VdW)).

[0083] Where N is the number of qualifying interactions for a specificterm and, in one embodiment, ΔG₀, ΔG_(hb), ΔG_(ionic), ΔG_(lipo) andΔG_(rot) are constants which are given the values: 5.4, −4.7, −4.7,−0.17, and 1.4, respectively.

[0084] The term N_(hb) is calculated according to equation 2:

N _(hb)=Σ_(h-bonds) f(ΔR, Δα)×f(N _(neighb))×f _(pcs)

[0085] f(ΔR, Δα) is a penalty function which accounts for largedeviations of hydrogen bonds from ideality and is calculated accordingto equation 3:

f(ΔR, Δ−α)=f1(ΔR)×f2(Δα)

Where:

f1(ΔR)=1 if ΔR<=TOL

or =1−(ΔR−TOL)/0.4 if ΔR<=0.4+TOL

or =0 if ΔR>0.4+TOL

And:

f2(Δα)=1 if Δα<30°

or =1−(Δα−30)/50 if Δα<=80°

or =0 if Δα>80°

[0086] TOL is the tolerated deviation in hydrogen bond length=0.25 Å

[0087] ΔR is the deviation of the H—O/N hydrogen bond length from theideal value=1.9 Å

[0088] Δα is the deviation of the hydrogen bond angle∠_(N/O—H . . . O/N) from its idealized value of 180°

[0089] f(N_(neighb)) distinguishes between concave and convex parts of aprotein surface and therefore assigns greater weight to polarinteractions found in pockets rather than those found at the proteinsurface. This function is calculated according to equation 4 below:

f(N _(neighb))=(N _(neighb) /N _(neighb,0))^(α) where α=0.5

[0090] N_(neighb) is the number of non-hydrogen protein atoms that arecloser than 5 Å to any given protein atom.

[0091] N_(neighb,0) is a constant=25

[0092] f_(pcs) is a function which allows for the polar contact surfacearea per hydrogen bond and therefore distinguishes between strong andweak hydrogen bonds and its value is determined according to thefollowing criteria:

f_(pcs)=β when A_(polar)/N_(HB)<10 Å²

or f_(pcs)=1 when A_(polar)/N_(HB)>10 Å²

[0093] A_(polar) is the size of the polar protein-ligand contact surface

[0094] N_(HB) is the number of hydrogen bonds

[0095] β is a constant whose value=1.2

[0096] For the implementation of the modified Böhm scoring function, thecontributions from ionic interactions, ΔG_(ionic), are computed in asimilar fashion to those from hydrogen bonds described above since thesame geometry dependency is assumed.

[0097] The term N_(lipo) is calculated according to equation 5 below:

N _(lipo)=Σ_(1L) f(r _(1L))

[0098] f(r_(1L)) is calculated for all lipophilic ligand atoms, l, andall lipophilic protein atoms, L, according to the following criteria:

f(r _(1L))=1 when r _(1L) <=R1f(r _(1L))=(r _(1L) −R1)/(R2−R1) when R2<r_(1L) >R1

f(r _(1L))=0 when r _(1L) >=R2

Where:

R1=r ₁ ^(vdw) +r _(L) ^(vdw)+0.5

and R2=R1+3.0

[0099] and r₁ ^(vdw) is the Van der Waal's radius of atom l

[0100] and r_(L) ^(vdw) is the Van der Waal's radius of atom L

[0101] The term N_(rot) is the number of rotable bonds of the amino acidside chain and is taken to be the number of acyclic sp³-sp³ and sp³-sp²bonds. Rotations of terminal —CH₃ or —NH₃ are not taken into account.

[0102] The final term, E_(VdW), is calculated according to equation 6below:

E _(VdW)=ε₁ε₂((r ₁ ^(vdw) +r ₂ ^(vdw))¹² /r ¹²−(r ₁ ^(vdw) +r ₂ ^(vdw))⁶/r ⁶), where:

[0103] ε₁ and ε₂ are constants dependant upon atom identity

[0104] r₁ ^(vdw)+r₂ ^(vdw) are the Van der Waal's atomic radii

[0105] r is the distance between a pair of atoms.

[0106] With regard to Equation 6, in one embodiment, the constants ε₁and ε₂ are given the atom values: C: 0.245, N: 0.283, O: 0.316, S:0.316, respectively (i.e. for atoms of Carbon, Nitrogen, Oxygen andSulphur, respectively). With regards to equations 5 and 6, the Van derWaal's radii are given the atom values C: 1.85, N: 1.75, O: 1.60, S:2.00 Å.

[0107] It should be understood that all predetermined values andconstants given in the equations above are determined within theconstraints of current understandings of protein ligand interactionswith particular regard to the type of computation being undertakenherein. Therefore, it is possible that, as this scoring function isrefined further, these values and constants may change hence anysuitable numerical value which gives the desired results in terms ofestimating the binding energy of a protein to a ligand may be used andhence fall within the scope of the present invention. As describedabove, the scoring function is applied to data extracted from thedatabase of side-chain conformations, atom identities, and interatomicdistances. For the purposes of the present description, the number ofMHC Class II molecules included in this database is 42 models plus foursolved structures. It should be apparent from the above descriptionsthat the modular nature of the construction of the computational methodof the present invention means that new models can simply be added andscanned with the peptide backbone library and side-chain conformationalsearch function to create additional data sets which can be processed bythe peptide scoring function as described above. This allows for therepertoire of scanned MHC Class II molecules to easily be increased, orstructures and associated data to be replaced if data are available tocreate more accurate models of the existing alleles. The presentprediction method can be calibrated against a data set comprising alarge number of peptides whose affinity for various MHC Class IImolecules has previously been experimentally determined. By comparisonof calculated versus experimental data, a cut of value can be determinedabove which it is known that all experimentally determined T-cellepitopes are correctly predicted. It should be understood that, althoughthe above scoring function is relatively simple compared to somesophisticated methodologies that are available, the calculations areperformed extremely rapidly. It should also be understood that theobjective is not to calculate the true binding energy per se for eachpeptide docked in the binding groove of a selected MHC Class II protein.The underlying objective is to obtain comparative binding energy data asan aid to predicting the location of T-cell epitopes based on theprimary structure (i.e. amino acid sequence) of a selected protein. Arelatively high binding energy or a binding energy above a selectedthreshold value would suggest the presence of a T-cell epitope in theligand. The ligand may then be subjected to at least one round ofamino-acid substitution and the binding energy recalculated. Due to therapid nature of the calculations, these manipulations of the peptidesequence can be performed interactively within the program's userinterface on cost-effectively available computer hardware. Majorinvestment in computer hardware is thus not required. It would beapparent to one skilled in the art that other available software couldbe used for the same purposes. In particular, more sophisticatedsoftware which is capable of docking ligands into protein binding-sitesmay be used in conjunction with energy minimization. Examples of dockingsoftware are: DOCK (Kuntz et al., J. Mol. Biol., 161:269-288 (1982)),LUDI (Böhm, H. J., J. Comput Aided Mol. Des., 8:623-632 (1994)) andFLEXX (Rarey M., et al., ISMB, 3:300-308 (1995)). Examples of molecularmodeling and manipulation software include: AMBER (Tripos) and CHARMm(Molecular Simulations Inc.). The use of these computational methodswould severely limit the throughput of the method of this invention dueto the lengths of processing time required to make the necessarycalculations. However, it is feasible that such methods could be used asa ‘secondary screen’ to obtain more accurate calculations of bindingenergy for peptides which are found to be ‘positive binders’ via themethod of the present invention. The limitation of processing time forsophisticated molecular mechanic or molecular dynamic calculations isone which is defined both by the design of the software which makesthese calculations and the current technology limitations of computerhardware. It may be anticipated that, in the future, with the writing ofmore efficient code and the continuing increases in speed of computerprocessors, it may become feasible to make such calculations within amore manageable time-frame. Further information on energy functionsapplied to macromolecules and consideration of the various interactionsthat take place within a folded protein structure can be found in:Brooks, B. R., et al., J. Comput. Chem., 4:187-217 (1983) and furtherinformation concerning general protein-ligand interactions can be foundin: Dauber-Osguthorpe et al., Proteins4(1):31-47(1988), which areincorporated herein by reference in their entirety. Useful backgroundinformation can also be found, for example, in Fasman, G. D., ed.,Prediction of Protein Structure and the Principles of ProteinConformation, Plenum Press, New York, ISBN: 0-306 4313-9.

1 50 1 127 PRT Homo Sapiens 1 Ala Pro Ala Arg Ser Pro Ser Pro Ser ThrGln Pro Trp Glu His Val 1 5 10 15 Asn Ala Ile Gln Glu Ala Arg Arg LeuLeu Asn Leu Ser Arg Asp Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr Val GluVal Ile Ser Glu Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu Gln ThrArg Leu Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys LeuLys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln His CysPro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Thr Ile Thr Phe GluSer Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu Leu Val Ile Pro PheAsp Cys Trp Glu Pro Val Gln Glu 115 120 125 2 13 PRT Artificial SequenceHMC Class II binding epitope 2 Pro Ser Pro Ser Thr Gln Pro Trp Glu HisVal Asn Ala 1 5 10 3 13 PRT Artificial Sequence HMC Class II bindingepitope 3 Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg 1 5 10 413 PRT Artificial Sequence HMC Class II binding epitope 4 Glu His ValAsn Ala Ile Gln Glu Ala Arg Arg Leu Leu 1 5 10 5 13 PRT ArtificialSequence HMC Class II binding epitope 5 His Val Asn Ala Ile Gln Glu AlaArg Arg Leu Leu Asn 1 5 10 6 13 PRT Artificial Sequence HMC Class IIbinding epitope 6 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu 15 10 7 13 PRT Artificial Sequence HMC Class II binding epitope 7 Asn AlaIle Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser 1 5 10 8 13 PRT ArtificialSequence HMC Class II binding epitope 8 Arg Arg Leu Leu Asn Leu Ser ArgAsp Thr Ala Ala Glu 1 5 10 9 13 PRT Artificial Sequence HMC Class IIbinding epitope 9 Arg Leu Leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met 15 10 10 13 PRT Artificial Sequence HMC Class II binding epitope 10 LeuAsn Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu 1 5 10 11 13 PRTArtificial Sequence HMC Class II binding epitope 11 Arg Asp Thr Ala AlaGlu Met Asn Glu Thr Val Glu Val 1 5 10 12 13 PRT Artificial Sequence HMCClass II binding epitope 12 Ala Glu Met Asn Glu Thr Val Glu Val Ile SerGlu Met 1 5 10 13 13 PRT Artificial Sequence HMC Class II bindingepitope 13 Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp Leu 1 5 10 1413 PRT Artificial Sequence HMC Class II binding epitope 14 Glu Thr ValGlu Val Ile Ser Glu Met Phe Asp Leu Gln 1 5 10 15 13 PRT ArtificialSequence HMC Class II binding epitope 15 Val Glu Val Ile Ser Glu Met PheAsp Leu Gln Glu Pro 1 5 10 16 13 PRT Artificial Sequence HMC Class IIbinding epitope 16 Glu Val Ile Ser Glu Met Phe Asp Leu Gln Glu Pro Thr 15 10 17 13 PRT Artificial Sequence HMC Class II binding epitope 17 IleSer Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu 1 5 10 18 13 PRTArtificial Sequence HMC Class II binding epitope 18 Ser Glu Met Phe AspLeu Gln Glu Pro Thr Cys Leu Gln 1 5 10 19 13 PRT Artificial Sequence HMCClass II binding epitope 19 Glu Met Phe Asp Leu Gln Glu Pro Thr Cys LeuGln Thr 1 5 10 20 13 PRT Artificial Sequence HMC Class II bindingepitope 20 Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg 1 5 10 2113 PRT Artificial Sequence HMC Class II binding epitope 21 Phe Asp LeuGln Glu Pro Thr Cys Leu Gln Thr Arg Leu 1 5 10 22 13 PRT ArtificialSequence HMC Class II binding epitope 22 Glu Pro Thr Cys Leu Gln Thr ArgLeu Glu Leu Tyr Lys 1 5 10 23 13 PRT Artificial Sequence HMC Class IIbinding epitope 23 Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln Gly 15 10 24 13 PRT Artificial Sequence HMC Class II binding epitope 24 GlnThr Arg Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly 1 5 10 25 13 PRTArtificial Sequence HMC Class II binding epitope 25 Thr Arg Leu Glu LeuTyr Lys Gln Gly Leu Arg Gly Ser 1 5 10 26 13 PRT Artificial Sequence HMCClass II binding epitope 26 Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly SerLeu Thr 1 5 10 27 13 PRT Artificial Sequence HMC Class II bindingepitope 27 Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys 1 5 10 2813 PRT Artificial Sequence HMC Class II binding epitope 28 Gln Gly LeuArg Gly Ser Leu Thr Lys Leu Lys Gly Pro 1 5 10 29 13 PRT ArtificialSequence HMC Class II binding epitope 29 Arg Gly Ser Leu Thr Lys Leu LysGly Pro Leu Thr Met 1 5 10 30 13 PRT Artificial Sequence HMC Class IIbinding epitope 30 Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met 15 10 31 13 PRT Artificial Sequence HMC Class II binding epitope 31 SerLeu Thr Lys Leu Lys Gly Pro Leu Thr Met Met Ala 1 5 10 32 13 PRTArtificial Sequence HMC Class II binding epitope 32 Thr Lys Leu Lys GlyPro Leu Thr Met Met Ala Ser His 1 5 10 33 13 PRT Artificial Sequence HMCClass II binding epitope 33 Lys Gly Pro Leu Thr Met Met Ala Ser His TyrLys Gln 1 5 10 34 13 PRT Artificial Sequence HMC Class II bindingepitope 34 Gly Pro Leu Thr Met Met Ala Ser His Tyr Lys Gln His 1 5 10 3513 PRT Artificial Sequence HMC Class II binding epitope 35 Pro Leu ThrMet Met Ala Ser His Tyr Lys Gln His Cys 1 5 10 36 13 PRT ArtificialSequence HMC Class II binding epitope 36 Leu Thr Met Met Ala Ser His TyrLys Gln His Cys Pro 1 5 10 37 13 PRT Artificial Sequence HMC Class IIbinding epitope 37 Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro 15 10 38 13 PRT Artificial Sequence HMC Class II binding epitope 38 SerHis Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr 1 5 10 39 13 PRTArtificial Sequence HMC Class II binding epitope 39 Cys Pro Pro Thr ProGlu Thr Ser Cys Ala Thr Gln Thr 1 5 10 40 13 PRT Artificial Sequence HMCClass II binding epitope 40 Pro Glu Thr Ser Cys Ala Thr Gln Thr Ile ThrPhe Glu 1 5 10 41 13 PRT Artificial Sequence HMC Class II bindingepitope 41 Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu 1 5 10 4213 PRT Artificial Sequence HMC Class II binding epitope 42 Gln Thr IleThr Phe Glu Ser Phe Lys Glu Asn Leu Lys 1 5 10 43 13 PRT ArtificialSequence HMC Class II binding epitope 43 Ile Thr Phe Glu Ser Phe Lys GluAsn Leu Lys Asp Phe 1 5 10 44 13 PRT Artificial Sequence HMC Class IIbinding epitope 44 Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe Leu Leu Val 15 10 45 13 PRT Artificial Sequence HMC Class II binding epitope 45 SerPhe Lys Glu Asn Leu Lys Asp Phe Leu Leu Val Ile 1 5 10 46 13 PRTArtificial Sequence HMC Class II binding epitope 46 Glu Asn Leu Lys AspPhe Leu Leu Val Ile Pro Phe Asp 1 5 10 47 13 PRT Artificial Sequence HMCClass II binding epitope 47 Asn Leu Lys Asp Phe Leu Leu Val Ile Pro PheAsp Cys 1 5 10 48 13 PRT Artificial Sequence HMC Class II bindingepitope 48 Lys Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu 1 5 10 4913 PRT Artificial Sequence HMC Class II binding epitope 49 Asp Phe LeuLeu Val Ile Pro Phe Asp Cys Trp Glu Pro 1 5 10 50 13 PRT ArtificialSequence HMC Class II binding epitope 50 Leu Leu Val Ile Pro Phe Asp CysTrp Glu Pro Val Gln 1 5 10

1. A modified molecule having the biological activity of humangranulocyte macrophage colony stimulating factor (GM-CSF) and beingsubstantially non-immunogenic or less immunogenic than any non-modifiedmolecule having the same biological activity when used in vivo.
 2. Amolecule according to claim 1, wherein said loss of immunogenicity isachieved by removing one or more T-cell epitopes derived from theoriginally non-modified molecule.
 3. A molecule according to claim 1 or2, wherein said loss of immunogenicity is achieved by reduction innumbers of MHC allotypes able to bind peptides derived from saidmolecule.
 4. A molecule according to claim 2 or 3, wherein one T-cellepitope is removed.
 5. A molecule according to any of the claims 2-4,wherein said originally present T-cell epitopes are MHC class II ligandsor peptide sequences which show the ability to stimulate or bind T-cellsvia presentation on class II.
 6. A molecule according to claim 5,wherein said peptide sequences are selected from the group as depictedin Table
 1. 7. A molecule according to any of the claims 2-6, wherein1-9 amino acid residues in any of the originally present T-cell epitopesare altered.
 8. A molecule according to claim 7, wherein one amino acidresidue is altered.
 9. A molecule according to claim 7 or 8, wherein thealteration of the amino acid residues is substitution of originallypresent amino acid(s) residue(s) by other amino acid residue(s) atspecific position(s).
 10. A molecule according to claim 9, wherein oneor more of the amino acid residue substitutions are carried out asindicated in Table
 2. 11. A molecule according to claim 10, whereinadditionally one or more of the amino acid residue substitutions arecarried out as indicated in Table 3 for the reduction in the number ofMHC allotypes able to bind peptides derived from said molecule.
 12. Amolecule according to claim 9, wherein one or more amino acidsubstitutions are carried as indicated in Table
 3. 13. A moleculeaccording to claim 7 or 8, wherein the alteration of the amino acidresidues is deletion of originally present amino acid(s) residue(s) atspecific position(s).
 14. A molecule according to claim 7 or 8, whereinthe alteration of the amino acid residues is addition of amino acid(s)at specific position(s) to those originally present.
 15. A moleculeaccording to any of the claims 7 to 14, wherein additionally furtheralteration is conducted to restore biological activity of said molecule.16. A molecule according to claim 15, wherein the additional furtheralteration is substitution, addition or deletion of specific aminoacid(s).
 17. A modified molecule according to any of the claims 7-16,wherein the amino acid alteration is made with reference to anhomologous protein sequence.
 18. A modified molecule according to any ofthe claims 7-16, wherein the amino acid alteration is made withreference to in silico modeling techniques.
 19. A DNA sequence codingfor a modified GM-CSF of any of the claims 1-18.
 20. A pharmaceuticalcomposition comprising a modified molecule having the biologicalactivity of GM-CSF as defined in any of the above-cited claims,optionally together with a pharmaceutically acceptable carrier, diluentor excipient.
 21. A method for manufacturing a modified molecule havingthe biological activity of GM-CSF as defined in any of the claims of theabove-cited claims comprising the following steps: (i) determining theamino acid sequence of the polypeptide or part thereof. (ii) identifyingone or more potential T-cell epitopes within the amino acid sequence ofthe protein by any method including determination of the binding of thepeptides to MHC molecules using in vitro or in silico techniques orbiological assays; (iii) designing new sequence variants with one ormore amino acids within the identified potential T-cell epitopesmodified in such a way to substantially reduce or eliminate the activityof the T-cell epitope as determined by the binding of the peptides toMHC molecules using in vitro or in silico techniques or biologicalassays, or by binding of peptide-MHC complexes to T-cells; (iv)constructing such sequence variants by recombinant DNA techniques andtesting said variants in order to identify one or more variants withdesirable properties; and (v) optionally repeating steps (ii)-(iv). 22.A method of claim 21, wherein step (iii) is carried out by substitution,addition or deletion of 1-9 amino acid residues in any of the originallypresent T-cell epitopes.
 23. A method of claim 22, wherein thealteration is made with reference to a homologues protein sequenceand/or in silico modeling techniques.
 24. A method of any of the claims21-23, wherein step (ii) is carried out by the following steps: (a)selecting a region of the peptide having a known amino acid residuesequence; (b) sequentially sampling overlapping amino acid residuesegments of predetermined uniform size and constituted by at least threeamino acid residues from the selected region; (c) calculating MHC ClassII molecule binding score for each said sampled segment by summingassigned values for each hydrophobic amino acid residue side chainpresent in said sampled amino acid residue segment; and (d) identifyingat least one of said segments suitable for modification, based on thecalculated MHC Class II molecule binding score for that segment, tochange overall MHC Class II binding score for the peptide withoutsubstantially the reducing therapeutic utility of the peptide.
 25. Amethod of claim 24, wherein step (c) is carried out by using a Böhmscoring function modified to include 12-6 van der Waal's ligand-proteinenergy repulsive term and ligand conformational energy term by (1)providing a first data base of MHC Class II molecule models; (2)providing a second data base of allowed peptide backbones for said MHCClass II molecule models; (3) selecting a model from said first database; (4) selecting an allowed peptide backbone from said second database; (5) identifying amino acid residue side chains present in eachsampled segment; (6) determining the binding affinity value for all sidechains present in each sampled segment; and repeating steps (1) through(5) for each said model and each said backbone.
 26. A 13mer T-cellepitope peptide having a potential MHC class II binding activity andcreated from non-modified GM-CSF, selected from the group as depicted inTable
 1. 27. A peptide sequence consisting of at least 9 consecutiveamino acid residues of a 13mer T-cell epitope peptide according to claim26.
 28. Use of a 13mer T-cell epitope peptide according to claim 26 forthe manufacture of GM-CSF having substantially no or less immunogenicitythan any non-modified molecule with the same biological activity whenused in vivo.
 29. Use of a peptide sequence according to claim 27 forthe manufacture of GM-CSF having substantially no or less immunogenicitythan any non-modified molecule with the same biological activity whenused in vivo.